The Duffy blood group system consists of two major antigens Fya and Fyb produced by FY+A and FY+B co-dominant alleles. Antisera, anti-Fya and anti-Fyb define four phenotypes, Fy(a+b-), Fy(a-b+), Fy(a+b+), and Fy(a-b- ). Neither antiserum agglutinates Duffy Fy(a-b-) cells, the predominant phenotype in Blacks. Blacks with Fy(a-b-) erythrocytes cannot be infected by the human malarial parasite P. vivax and simian parasite P. knowlesi. We cloned the Duffy gene and showed that it encodes a glycoprotein (gp-Fy) of 337 residues. The objectives of this proposal are: (1) To determine the topology of the N- and C-terminal domains of gp-Fy, which will be done either by immunochemical binding of whole erythrocytes and inside-out membrane vesicles or double labeling immunoelectromicroscopy. (2) The determination, at the N-terminal domain, of the amino acids necessary for recognition sites of antibodies and malarial parasites. This will be carried out with ELISA assay of chemically synthesized peptides, followed by the construction of amino acid deletion and substitution mutants and expression in K562 cells. (3) The sequencing of non-erythroid gp-Fy mRNA in kidney, lung, thymus, spleen and brain. (4) The characterization by immunocytochemistry, of which cell(s) in these tissues produce gp-Fy. (5) Identification of the major ligand-binding requirements for chemokine recognition. This will be done by the construction of a panel of substitution and deletion mutants and expression in K562 cells. (6) The search for homologous FY in mouse will be done in a genomic library that will be screened with a probe containing either bone marrow cDNA or a probe having bone marrow and brain cDNA sequences. (7) To study interactions of Duffy and Rh protein since a Duffy antigenic determinant (Fy5) depends upon the presence of the Rh protein. The study will be done in Duffy-transfected K562 cells. The long-term goals of this project are to determine: (1) the gp-Fy protein function(s); (2) the significance of the Duffy-like protein in brain cells; (3) the sequence conformation that binds to the merozoites and the design of drugs which would block invasion; (4) whether gp-Fy protein is a transporter and finally, (5) the design of mouse knockout experiments to determine the significance of Duffy gene. This research project will have major biomedical consequences in the design of more sensitive and reliable reagents for the detection of Duffy antigens, anticipation of hemolytic diseases in the newborn, design of drugs to prevent parasite invasion, defining gp-Fy role as the human erythrocyte chemokine receptor and unraveling the function of Duffy gene. The Duffy gene is active in a variety of tissues, and these studies will establish its relevance in human biology.